Inhibition of Calcium Dependent Protein Kinase 1 (CDPK1) by Pyrazolopyrimidine Analogs Decreases Establishment and Reoccurrence of Central Nervous System Disease by Toxoplasma gondii

Article abstract of DOI:10.1021/acs.jmedchem.7b01192

Calcium dependent protein kinase 1 (CDPK1) is an essential enzyme in the opportunistic pathogen Toxoplasma gondii. CDPK1 controls multiple processes that are critical to the intracellular replicative cycle of T. gondii including secretion of adhesins, motility, invasion, and egress. Remarkably, CDPK1 contains a small glycine gatekeeper residue in the ATP binding pocket making it sensitive to ATP-competitive inhibitors with bulky substituents that complement this expanded binding pocket. Here we explored structure-activity relationships of a series of pyrazolopyrimidine inhibitors of CDPK1 with the goal of increasing selectivity over host enzymes, improving antiparasite potency, and improving metabolic stability. The resulting lead compound 24 exhibited excellent enzyme inhibition and selectivity for CDPK1 and potently inhibited parasite growth in vitro. Compound 24 was also effective at treating acute toxoplasmosis in the mouse, reducing dissemination to the central nervous system, and decreasing reactivation of chronic infection in severely immunocompromised mice. These findings provide proof of concept for the development of small molecule inhibitors of CDPK1 for treatment of CNS toxoplasmosis.

Full text of DOI:10.1021/acs.jmedchem.7b01192

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Article
Inhibition of calcium dependent protein kinase 1 (CDPK1) by
pyrazolopyrimidine analogs decreases establishment and reoccurrence
of central nervous system disease by Toxoplasma gondii.
Florentine U. Rutaganira, Jennifer Barks, Mary Savari Dhason, Qiuling Wang, Michael
Steven Lopez, Shaojun Long, Joshua B Radke, Nathaniel G Jones, Amarendar R Maddirala,
James W Janetka, Majida El Bakkouri, Raymond Hui, Kevan M. Shokat, and L. David Sibley
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01192 • Publication Date (Web): 21 Sep 2017
Downloaded from http://pubs.acs.org on September 23, 2017
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Jmꢀ2017ꢀ01192g
Inhibition of calcium dependent protein kinase 1 (CDPK1) by
pyrazolopyrimidine analogs decreases establishment and
reoccurrence of central nervous system disease by Toxoplasma
gondii.
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2a
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Florentine U. Rutaganira , Jennifer Barks , Mary Savari Dhason , Qiuling Wang ,
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Michael S. Lopez , Shaojun Long , Joshua B. Radke , Nathaniel G. Jones , Amarendar
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R. Maddirala . James W. Janetka , Majida El Bakkouri , Raymond Hui , Kevan M.
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Shokat , and L. David Sibley .
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Howard Hughes Medical Institute and Department of Cellular and Molecular
Pharmacology, University of California, San Francisco, San Francisco, California
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4158, USA
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Department of Molecular Microbiology, Washington University School of Medicine, St.
Louis, MO, 63130, USA
Department of Biochemistry and Molecular Biophysics, Washington University School
of Medicine, St. Louis, MO, 63130, USA
Structural Genomics Consortium, University of Toronto, MaRS South Tower, 101
College St, Toronto, ON, M5G 1L7, Canada
Toronto General Hospital Research Institute, 200 Elizabeth St., Toronto, ON M5G
2C4, Canada
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contributed equally, listed alphabetically,
Achaogen Inc. 1 Tower Place, #300, South San Francisco, CA 94080
Corresponding author
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Key words: Parasite, opportunistic pathogen, chronic infection, tissue cyst, bradyzoite,
encephalitis, AIDS, protein kinase, ATPꢀbinding pocket, structure activity relationship,
pharmacokinetics, Xꢀray crystal structure,
ABSTRACT: Calcium dependent protein kinase 1 (CDPK1) is an essential enzyme in
the opportunistic pathogen Toxoplasma gondii. CDPK1 controls multiple processes that
are essential to the intracellular replicative cycle of T. gondii including secretion of
adhesins, motility, invasion, and egress. Remarkably, CDPK1 contains a small glycine
gatekeeper residue in the ATP binding pocket making it sensitive to ATPꢀcompetitive
inhibitors with bulky substituents that complement this expanded binding pocket. Here
we explored structureꢀactivity relationships of a series of pyrazolopyrimidine inhibitors of
CDPK1 with the goals of increasing selectivity over host enzymes, improving antiꢀ
parasite potency, and improving metabolic stability. The resulting lead compound 24
exhibits excellent enzyme inhibition and selectivity for CDPK1 and potently inhibited
parasite growth in vitro. Compound 24 was also effective at treating acute
toxoplasmosis in the mouse, reducing dissemination to the central nervous system,
decreasing reactivation of chronic infection in severely immunocompromised mice.
These findings provide proof of concept for the development of small molecule inhibitors
of CDPK1 for treatment of CNS toxoplasmosis.
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INTRODUCTION
Toxoplasma gondii is a widespread protozoan parasite of animals that causes zoonotic
infections in humans. Although most human cases are well controlled, infection in
immunocompromised patients leads to serious sequelae, including toxoplasmic
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encephalitis and pneumonia, which are lifeꢀthreatening if not treated . Although the
advent of HAART therapy has reduced the frequency of toxoplasmosis as an
opportunistic pathogen in developed countries, it is still a serious complication in many
parts of the world where patients do not have adequate access to testing or treatment
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for HIV infection . Additionally, toxoplasmosis can cause serious problems in organ
transplant and cancer chemotherapy patients due to their immunocompromised status
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.
Furthermore, toxoplasmosis is a recognized cause of severe ocular disease in
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healthy adults in some locations such as Brazil .
Current therapy for toxoplasmosis is based on combination of pyrimethamine, which
blocks dihydrofolate reductase (DHFR), and sulfadiazine, a folate antagonist.
Collectively these drugs disrupt replication by inhibiting nucleic acid synthesis. This
drug combination is efficacious in treating acute infection through blocking replication of
tachyzoites. Unfortunately pyrimethamine is associated with several adverse side
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effects including anemia due to bone marrow suppression and many patients
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experience allergic reactions to sulfonamide drugs . Chronic infections caused by T.
gondii are typified by slow growing bradyzoites that reside within thickꢀwalled tissue
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cysts . The emergence of bradyzoites upon cyst rupture is thought to give rise to
daughter cysts that sustain the chronic infection and contribute to recurrence of actively
replicating tachyzoites when the parasite reverts to the lytic form. Hence, treatments
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that could block reꢀemergence from the tissue cyst, or block invasion of host cells by
bradyzoites could interrupt this cycle and eliminate chronic infection. Unfortunately,
current therapies that inhibit DHFR and antagonize the folate pathway are not effective
at clearing chronic infection, as evidenced by the high relapse in immunocompromised
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patients when therapy is discontinued , presumably due to the slow and sporadic
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replication of bradyzoites .
One of the key steps in defining new leads for therapeutic intervention is to identify
essential pathways that can be targeted by small molecules. One potential new target
is that fulfills these criteria is calcium dependent protein kinase 1 (CDPK1) in T. gondii
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.
Genetic depletion or chemical inhibition of CDPK1 blocks adhesion secretion and
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compromises motility, cell invasion, and egress , thus demonstrating the essentiality of
this target. The Xꢀray crystal structure of CDPK1 also revealed an unusual feature in
the ATPꢀbinding pocket where a glycine (G) residue occupies the gatekeeper position
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where there is normally a larger hydrophobic residue
. The presence of G
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gatekeeper is entirely unprecedented in human kinases , and CDPK1 is the only
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kinase in T. gondii with this unusual feature . As a consequence, CDPK1 is
exquisitely sensitive to bulky ATP competitive inhibitors such as pyrazolopyrimidines
(PP), which mimic the nucleotide binding interactions with the hinge region within the
ATPꢀbinding pocket and project bulky substituents into the expanded hydrophobic
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pocket created by the G gatekeeper
. These features have been exploited to
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develop PP analogs that are potent inhibitors of CDPK1 in T. gondii
, and the most
promising of these compounds show good efficacy in mouse models of toxoplasmosis
22ꢀ24
.
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Our prior studies involving derivatives with substituents (R1) to the C3 position of
the pyrazolopyrimdine (PP) core revealed the importance of the methylene linkage at
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C3 to confer flexibility of R1 substituent binding . However, compounds with the
methylene linkage overall were not stable in the presence of rat liver microsomes,
potentially due to the methylene bridge being subject to cytochrome P450 metabolism.
To address these deficiencies, we have more fully explored the design and synthesis of
different heteroatom linkages at the C3 position and expanded the diversity of
substituents at the R2 position. Collectively, these rationally driven modifications have
identified new TgCDPK1 inhibitors with excellent potency in addition to improved
selectivity, metabolic stability, and in vivo efficacy in controlling toxoplasmosis in the
mouse.
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RESULTS
Design and synthesis of new PP analogs targeting CDPK1
To identify compounds with improved metabolic stability that would also exploit
structural differences between CDPK1 and hSrc active sites, we designed and
synthesized a compound library containing varied functional groups at R1 attached to
the C3 position of the PP core (Scheme 1). These were either linked to the core
pyrazolopyrimidine or pyrrolopyrimidine scaffold via methylene or heteroatom linkages
(X) in combination with tꢀbutyl or various R2 groups at the N1 position (Scheme 1). The
hSrc kinase was chosen for initial counterꢀscreening because it contains a threonine (T)
gatekeeper residue that represents one of the smallest amino acids found in native
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human kinases . All compounds were synthesized using previously described
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methods to form the pyrazolopyrimidine scaffold or with advanced pyrrolopyrimidine
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intermediates
. Methods to generate compounds with methylene linkage at C3
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have been previously reported and heteroatom linkages were incorporated using
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established aryl coupling chemistry
(Scheme 1). Detailed procedures for chemical
synthesis and compound characterization are presented in the supplementary
information.
Analysis of new PP analogs modified at the R1 position
To develop a structureꢀactivity relationship (SAR) profile, we investigated the effects of
modifying the R1 group and different linkers to the core scaffold at position C3 in a
series of compounds that contained tertiary butyl at the N1 position (Figure 1A). We
tested compounds for their potency against CDPK1 in vitro, using an ELISA assay for
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phosphorylated substrate, described previously . In parallel, we tested the inhibitors
for their ability to inhibit parasite growth in vitro using a βꢀGal expressing line of T. gondii
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to determine EC values, as described previously . Finally, we screened each analog
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for stability in vitro in the presence of rat liver microsomes, as a surrogate for estimating
in vivo metabolic stability.
Starting with parent compound 1, replacement of the C3 methylene linker with an
ether, thioether, or amine linkage improved metabolic stability for ether and amine
linkages (Figure 1A). This result confirms XenoSite in silico predictions of metabolism
at the methylene as in compound 1, that is less likely with ether and amine linkages as
in compounds 2 and 4 but is still possible with thioether linkages as in 3
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(Supplementary Figure S1) . The thioether linkage was also associated with
substantial loss of activity in the parasite inhibition assay (Figure 1A). We also
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observed increased metabolic stability of the ether linkage over methylene for another
compound set consisting of compounds 5, 6. Analogs bearing halogen substitutions
were generally more stable, especially in the presence of the ether linkage. The
addition of some ꢀmeta substituents on the benzyl ring was associated with loss of
metabolic stability, despite having an ether linkage, for example addition of the methoxy
group in compound 9 and the methyl group for compound 10 (Figure 1A). Since the
ether linkage generally improved metabolic stability while maintaining efficacy against
parasite replication, we selected ether linkages for further optimization.
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Structural interactions between PP compounds and CDPK1
To better understand the binding interactions of PP analogs and the CDPK1
enzyme, we solved a series of high–resolution coꢀcrystal Xꢀray structures, using
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previously described methods . The coꢀcrystal structure of CDPK1 with the lead
compound 1 revealed a characteristic hydrogenꢀbonding (Hꢀbonding) pattern of
interaction between the aminopyridine and the kinase hinge. More specifically, the
primary amine at C4 of the PP core acts as a Hꢀbond donor in an interaction with the
backbone carbonyl of glutamate (E) 129, and the N5 in the PP core acts as a Hꢀbond
acceptor in an interaction with the backbone amide of tyrosine (Y) 131 (Figure 1B).
Importantly, the coꢀcrystal structures of compounds 1 - 4 bound to CDPK1 show that
these modifications do not substantially change the binding conformations (Figure 1C).
Key Hꢀbonding interactions between the PP core and the hinge region are maintained
with R1 occupying the hydrophobic pocket formed by the presence of the small G
gatekeeper residue (Figure 1C).
Analysis of new PP analogs modified at the R2 position
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Previous studies of PP analogs have shown that larger substituents are tolerated at
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the R2 position and can lead to gain of potency and selectivity for CDPK1 . To
explore the size tolerance within the CDPK1 pocket at this region, we generated a
series of analogs with cyclic amines at R2 (Figure 2A). Structural studies have shown
that methyl piperidine in the R2 is positioned to form a Hꢀ bond with glutamate (E) 135
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in the ribose binding region of the ATP pocket of CDPK1
. A coꢀcrystal structure of
compound 13 confirmed this interaction is also present in the series studied here
(Figure 2B). The piperidine is predicted to be protonated under physiological
conditions and as shown in the coꢀcrystal structure, the additional H likely forms a salt
bridge with glutamine (E) 135 (Figure 2B). We explored a series of cyclic amines based
on piperidine where we elucidated the importance of proper placement and direction of
the amine to form the favorable chargeꢀcharge salt bridge interaction with E135. We
found that any deviation from the piperidine of 13 in this series abrogated CDPK1
activity and decreased inhibition of parasite replication in culture (Figure 2A).
Compounds 16 and 15, which contain a tetrahydropyran and pyridine group,
respectively, each significantly lost potency, emphasizing the key contribution of the Hꢀ
bond donor of the piperidine (Figure 2A). On the other hand, all cyclic amines showed
greatly enhanced stability in the rat liver microsome assay thus providing much support
for further R2 modification as an approach to improving metabolic stability (Figure 2A).
In another series, we explored the combination of methyl piperidine at R2 together
with the prior functional groups at R1 (Figure 3A). Consistent with findings above, the
methyl piperidine containing analogs generally showed improved metabolic stability in
comparison to their t ꢀbutyl counterparts (Figure 1A, 3A). For example, the presence of
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the methyl piperidine in compound 17 could impart stability despite having a methylene
linkage to C3 of the PP core (Figure 3A). Compounds with additional halogens in the
benzyl ring at R1 gained further increases in microsome stability (e.g. 21 and 22)
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(
Figure 3A). Furthermore, there were no significant differences between
pyrazolopyrimidine and pyrrolopyrimidine as the core scaffold as demonstrated by
compounds 18 vs. 23 (Figure 3A).
Despite the reproducible ability of methyl piperidine to improve microsome stability
throughout the entire series, this modification also resulted in reduced biochemical
potency and ability to block parasite replication. Hence, incorporation of methyl
piperidine at R2 results in a tradeoff between improved metabolic stability and loss in
potency. This effect may be explained by the larger size of these inhibitors that may not
be as easily accommodated in the CDPK binding pocket. The combination of ether
linked R1 as –mꢀCl with R2 piperidine as in compound 13 showed the largest
improvement over parent compound 1 while retaining reasonable activity in both
inhibition CDPK1 and parasite growth (Figure 3A). In contrast, methylation of the
piperidine nitrogen in compound 25 led to a loss in potency, further highlighting the
importance of the Hꢀbonding ability of the protonated amine (Figure 3A). Converting
the piperidine to a 6ꢀmembered ring δꢀlactam in compound 26 also lead to a loss in
enzyme potency and a complete loss in antiꢀparasitic activity (Figure 3A). We also
explored modifying the piperidine by addition of diꢀfluorine (diꢀF) ꢀmeta to the amine, as
3
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such modifications are known to favorably affect the properties of drugꢀlike molecules .
The diꢀF modification in compound 24 vs. 13, which both have etherꢀlinked R1
substitutions to the PP core, slightly improved enzyme inhibition and antiꢀparasitic
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activity (Figure 3A). A homology model of 24, based on the coꢀcrystal structure of
compound 13 with CDPK1, suggests that the piperidine amine is still in an appropriate
position to interact with E135 (Figure 3B, Table 1). We also made a similar
modification of diꢀF in a ꢀmeta position to the piperidine amine in compound 18 to
generate the compound 28, both of which have a thioether linkage connecting R1 to C3
in the PP core. Surprisingly in this case, the diꢀF modification reduced enzyme activity
and greatly diminished antiꢀparasitic activity. Modification of R2 to contain a
trifluormethylcyclopropyl moiety was also synthesized in an attempt to reduce metabolic
instability, based on a previous study showing this modification can improve stability of
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biaryl compounds relative to those containing tꢀbutyl . This compound 27 lost potency
Figure 3), suggesting it does not optimally fit in the CDPK1 ATP binding pocket,
(
moreover it did not improve stability, perhaps due to the methylene linkage of the
trifluormethylcyclopropyl moiety to N1 of the core.
Inhibition of CDPK1 in vitro correlates with parasite growth inhibition
To compare the potency of all of the new PP analogs synthesized here, we plotted the
log values for CDPK1 enzyme inhibition (IC ) vs. log values for parasite growth
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inhibition (EC ) (Figure 4A). Fitting these data with a linear regression analysis
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indicated a reasonable correlation (r = 0.58), supporting the conclusion that the activity
of these compounds against parasite growth stems largely from inhibition of CDPK1.
However, the fact that this correlation is not higher suggests that in vivo potency is also
affected by other factors including differences in cell permeability. Some inhibitors lie
above the diagonal line, which represents a perfect correlation, indicating they are less
potent in blocking parasite growth, perhaps due to differences in cell permeability or
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efflux (Figure 4A). Among the most potent inhibitors was the initial lead 1, and
derivatives that contain an oxygen linkage at R1 (e.g. 2 and 10), as well as those that
contain the methylꢀpiperidine at R2 (e.g. 13, 24)(Figure 4A). Based on a combination
of potency, selectivity and PK properties, we prioritized compounds and selected these
five analogs for further study. Other compounds that were potent in both assays were
deprioritized for lack of specificity (e.g. 4) or lack of stability (e.g. 9).
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Metabolic stability and cellular update of PP analogs
To help choose PP analogs most likely to be active in vivo, we broadly examined the
physical properties, metabolic stability in mouse liver microsomes, and cellular uptake of
a select group of PP analogs (Table1). All compounds have modest cLogP values,
consistent with their intermediate hydrophobicity, and are predicted to have relatively
small total polar surface area, properties that should facilitate penetration of the CNS
(Table 1). Consistent with the findings in rat liver microsomes, the ether linkage in
compound 2 reduced metabolic clearance in mouse liver microsomes when compared
to compound 1, although both values are still relatively high (Table 1). Compound 10,
which had similar clearance to compound 1 in rat liver microsomes despite having an
ether linkage, was more rapidly cleared in mouse liver microsomes (Table 1),
suggesting the methyl group at C3 might be prone to metabolism. Stability was further
improved when the R2 group was switched from tꢀbutyl to methyl piperidine as in
compounds 13 and 18. However these latter two compounds, which contain a basic
amine group that is charged a neutral pH, were also associated with substantial efflux in
MDCK cells (Table 1). In addition to the many other useful aspects of fluorine
substitutions in medicinal chemistry, in some contexts it has been shown to reduce the
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basicity of proximal amines by lowering the pKa . Accordingly, we modified the
piperidine ring by addition of diꢀfluorine (diꢀF), a modification reduced the pKa and nicely
eliminated the efflux problem (24 and 28; Table 1). However, metabolic stability was
diminished by the diꢀF modification in these compounds. Analogs such as 1 showed
high levels of plasma protein binding (PPB) (i.e. 99%), however this property was
greatly reduced for compounds containing methyl piperidine or the diꢀF methyl
piperidine (Table 1). Importantly, none of the PP analogs tested affected the growth of
human foreskin fibroblasts (HFF) cells in vitro at 10 ꢀM, indicating that they are not toxic
to host cells (Table 1).
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Analysis of target specificity of select PP analogs
To provide an assay for inhibitor selectivity we cloned a variant of human Src, which has
a threonine (T) residue at the gatekeeper (cꢀSrc allele), which provides for an
18
intermediate level of sensitivity to pyrazolopyrimidine compounds . Although the initial
lead compound 1 potently inhibited hSrc, modification of the R1 linkage to the PP core
via an ether or thioether at C3 led to greater selectivity (Figure 1A). Modification of R2
to include cyclic amines, including piperidine, also led to large increases in selectivity for
CDPK1 over hSrc (Figure 2A). Although Src has been used as a surrogate for human
19, 21
kinases with intermediate sensitivity to PP analogs
, it is only one member of a
diverse family of tyrosine kinases that differ substantially in their sensitivity to PP
35
inhibitors . As such, we profiled select inhibitors 1, 2, 13, and 24, which marked key
transitions in our SAR profile. We screened all four analogs at 1 ꢀM against a broad
panel of 489 wild type and diseaseꢀassociated human kinases (Figure 5). Inhibitor
selectivity increased along our SAR profile, starting from thirtyꢀnine kinases that are
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inhibited or displaced by compound 1 by over 50% to only one kinase (CK1ε) that was
inhibited or displaced by 24 by over 50% (Figure 5, Supplementary Table S2).
Although compounds 13 and 24 exhibited IC values of ≤ 100 nM at 2 ꢀM ATP, these
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values were increased almost 10 fold in the presence of 100 ꢀM ATP (Supplementary
Table S2). Additionally, we profiled an earlier inhibitor 3ꢀmethylꢀbenzylꢀPP (compound 1
23
in ) to compare our initial lead 1 in this prior series. We determined that the selectivity
profile for both of these compounds was similar (Supplementary Figure S3), while
selectivity was greatly increased for compounds 13 and 14. Full inhibitor profiling data
is provided in the supplementary information (Supplementary Tables S3-S5).
Potency of select PP analogs against ex vivo bradyzoites
Previous studies have emphasized the ability of PP analogs to block host cell invasion
19, 21, 23
and egress by the rapidly growing tachyzoite stage of T. gondii
. To examine the
ability of our PP inhibitors to block invasion of bradyzoites, we purified mature tissue
cysts from the brains of chronically infected mice and treated them in vitro with selected
PP inhibitors. We compared the effects of treatment for short intervals (i.e. 4 hr
followed by washꢀout and further culture in the absence of compound) vs. continuous
treatment during a 10ꢀ14 day plaquing assay. Consistent with previous reports showing
that treatment with pyrimethamine requires overnight treatment to be effective in
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blocking tachyzoite growth we observed that shortꢀterm treatment with pyrimethamine
had minimal effect on infection by bradyzoites while continuous treatment blocked
plaque formation (Figure 4B). In contrast, the PP inhibitors showed good (e.g. 13), or
excellent (e.g. 1, 24), ability to block infection following even shortꢀterm incubation
(Figure 4B). Because the compounds were only present during the initial interaction of
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the parasite with the host cell monolayer, we interpret these results to indicate that PP
inhibitors can block bradyzoite entry into host cells during the 4 hr treatment period,
resulting in greatly diminished plaque formation. Additionally, continual culture with PP
compounds resulted in greater inhibition (Figure 4B), consistent with its ability to block
the multiple rounds of invasion and egress that occur during plaque formation, although
it is likely that tachyzoites predominate during these longerꢀterm assays.
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Mouse pharmacokinetic studies
We examined the pharmacokinetics (PK) of several PP inhibitors in mice following
either i.v. injection or p.o. administration in mice. Compounds 1 and 2 showed
reasonable oral bioavailability (F) ranging from ~ 25ꢀ30%, Cmax levels, and moderate
clearance (Cl) values that were below the level of single pass circulation through the
liver in mouse (~90 ml/min/kg) (Table 2). Compound 10 showed a higher clearance
rate, but had similar halfꢀlife (T1/2) to 1 and 2 of ~ 1.5 to 2 hr (Table 2). However, this
compound suffered from poor oral bioavailability (Table 2). Although compound 13 had
greater in vitro stability in microsomes (Table 1), it also displayed rapid clearance in
vivo, perhaps due to efflux as indicated above. Compound 13 also showed lower Cmax
values and high tissue exposure as evidenced by its volume of distribution (Vss) (Table
2
). Finally, compound 24 showed the best combination of properties with good CMax
following oral dosing, moderate clearance rate, T1/2 ~1.5 hr, and very high oral
availability (e.g. ~ 94%) (Table 2). These collective properties of compound 24
provided the best PK profile and highest values for AUC, reflecting prolonged serum
exposure following oral dosing.
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Based on these favorable properties, we examined the PK of 24 in mice using
increasing oral doses from 10, 25, and 50 mg/kg (Figure 4C). At higher does, 24
showed a twoꢀphase decay curve in plasma, with rapid initial drop followed by a more
gradual plateau that was seen at 25 and 50 mg/kg (Figure 4C). Based on its protein
binding properties (12% free in serum), the concentration of compound 24 in serum
would need to be 2.08 ꢀM, for the free value to attain a level equivalent to the in vitro
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EC of ~0.250 ꢀM. This corresponds to a value of 856 ng/ml in serum, as shown by
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the red line in Figure 4C. The protein bound fraction in brain was slightly higher at
7.2%, or 2.8% free, and the ratio of total compound in the CNS vs. serum was 5.6, for
9
an effective brain vs. plasma ratio ~ 1.3. Consequently, we would expect that the brain
free concentration of compound 24 in the brain was approximately the same or slightly
higher than that shown for serum in Figure 4C.
Treatment of acute toxoplasmosis
Based on its favorable PK parameters, we tested compound 24 for its ability to prevent
toxoplasmosis in the mouse model. Following an initial loading dose of 50 mg/kg, 24
was administered orally at 25 mg/kg BID for 6 days, beginning 24 hr after challenge with
tachyzoites of the ME49 strain of T. gondii. Treatment with 24 decreased the frequency
of lethal infection (Figure 6A), and this was associated with reduced weight loss
(Figure 6B) and with decreased tissue burdens of the parasite, as revealed by
bioluminescent imaging (Figure 6C). Consistent with a decrease in acute burden, there
were fewer tissue cysts formed in the brains of chronically infected mice following
treatment with 24 (Figure 6D). The dramatic ability of 24 to decrease dissemination
was further shown by bioluminescent imaging, where control animals showed extensive
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expansion of the parasite with in the peritoneum at day 8 and spread to the head at day
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8 (Figure 6E,F). In contrast, treated mice were able to restrict the growth of the
parasite to the site of injection and did not show detectable signal in the head (Figure
E,F). Following treatment with 24, many animals showed tissue cyst levels below the
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levels of detection (40 cysts per brain) (Figure 6D). These animals were subsequently
bioꢀassayed by oral feeding of 20% of their brain homogenate into a naïve interferon
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gamma receptor knock out mouse (Ifngr1 ). Although 5 of 6 recipient animals
succumbed to infection, reflecting a low level of chronic infection in the donor, one
remained alive, indicating that compound 24 treatment had resulted in a complete cure
in this animal (Figure 6D, red triangle).
Treatment of reactivated CNS toxoplasmosis
To determine whether compound 24 could prevent reactivation of chronic
ꢀ/ꢀ
toxoplasmosis, we utilized mice lacking the IFNγ receptor (i.e. Ifngr1 ), which are highly
37
susceptible and rapidly succumb to infection . Treatment of such animals with
sulfadiazine will suppress the growth of tachyzoites, while allowing the development of
38
chronic tissue cysts . Subsequent removal of sulfadiazine results in rapid reactivation,
38
ꢀ/ꢀ
characterized by CNS encephalitis, pneumonia and death . We injected Ifngr1 mice
with a type II strain capable of causing chronic infection and treated them from day 2ꢀ22
with sulfadiazine in the drinking water, which prevented otherwise lethal infection.
Following removal of sulfadiazine (48 hr later), mice were treated with compound 24 at
a dose of 25 mg/kg or 40 mg/kg BID, or with vehicle only control, by oral gavage for 8
days (Figure 7A). Withdrawal of sulfadiazine from the control animals lead to rapid
reactivation of chronic infection led to death of the control mice within 10 days (Figure
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B). In contrast, treatment with compound 24 prolonged survival (Figure 7B). We also
monitored the progression infection using bioluminescence. At the beginning of the
treatment period, all of the animals were negative for bioluminescence, consistent with
the infection being chronic. Monitoring of the control animals revealed that reactivation
was first centered in the CNS, with later spread to the lungs prior to death (Figure 7C).
In contrast to the rapid expansion of parasites seen in control mice, the
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bioluminescence signal was muted in compound 24 treated mice, and reactivation was
significantly delayed (Figure 7C). Consistent with this delay in reactivation, compound
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4 treated mice survived significantly longer (Figure 7B). One animal treated with
compound 24 at 25 mg/kg and two animals treated at 40 mg/kg remained negative for
bioluminescence signals and survived for an additional 22 days after removal of
compound (red triangles in Figure 7B). Subsequent bioassay of brain homogenates
ꢀ/ꢀ
from these treated mice into naïve Ifngr1 mice did not result in transfer of infection,
confirming that these animals had been cured (Figure 7 B).
DISCUSSION
In previous work, we demonstrated that PP inhibitors containing –meta chloro benzyl
36
substituents at C3 of the PP core provide potent inhibition of CDPK1 in T gondii .
However, when coupled with isopropyl or tꢀbutyl at R2, these compounds suffer from
36
metabolic instability . Our profiling of host kinases here also reveals that compounds
like 1 lack specificity, and this is also likely true of other related derivatives that share
this basic architecture. To alleviate this problem, we altered the linkage connecting R1
to C3 from a methylene to a heteroatom consisting of O, S, or N. An ether linkage
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proved most optimal in increasing stability and selectivity, while compounds with a
thioether linkage were typically less active and compounds with an amine linkage were
less selective. The reasons for these differences are not exactly clear as coꢀcrystal
structures of these analogs showed very close overlap of the analogs in the binding
pocket. The lower activity of the thioether linkage may relate to the bulkier nature of this
atom within the linker, which is very close to the hinge region in the crystal structure.
We have previously shown that even subtle modification to this linker, such as
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substitution of fluorine for hydrogen, ablates activity .
By combining the ether linkage at C3 with a methyl piperidine at R2, we achieved
further gains in specificity, with only modest loss of potency. Other modifications to R2,
such as including a pyridine or methylation of the piperidine nitrogen, ablated activity
suggesting that the unsubstituted alkyl amine is critical for the potency. Indeed a coꢀ
crystal structure of compound 13 revealed this amine is important for forming a
hydrogen bond with glutamate (E) 135 in the riboseꢀbinding region, likely stabilizing the
interaction of the compound with the ATPꢀbinding pocket. Addition of the piperidine
group in the scaffold described here also resulted in dramatic improvement in specificity
over host kinases compared to the initial lead compounds 1 and 2. Compounds 13 and
2
4 only potently inhibited a single human kinase CK1ε under conditions tested here.
Surprisingly, CK1ε is also sensitive to other PP analogs including 1ꢀNMꢀPP1 and 1ꢀNAꢀ
PP1, despite its methionine (M) gatekeeper, which is normally a hallmark for resistance
39
to such analogs . The sensitivity of CKε to PP analogs has been attributed to an
unusual flexibility in the M sideꢀchain that allows access of bulky PP analogs into the
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ATPꢀbinding pocket . Further modification of the PP scaffold may result in inhibitors
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that would retain potency for CDPK1 over CKε. However, this off target activity may be
less of an issue in vivo since compound 24 retains good potency against parasite
growth in a cellꢀbased assay, while off target activity against CKε drops appreciably at
higher ATP levels.
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Despite the improvements in specificity achieved with the piperidine analogs, these
charged primary amines suffered from efflux problems in our study, likely due to the
34
high pKa . Addition of diꢀF in the piperidine ring decreased the predicted pKa and
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eliminated the efflux problem, consistent with previous examples in other scaffolds .
34
Previous experimental measurements of piperidine amines with fluoro substituents ,
combined with computational predictions of the compound 24 piperidine amine pKa
suggest that the piperidine amine in compound 24 is partially protonated at
physiological pH. Based on these properties and its potency, we would predict that
compound 24 is still able to form a Hꢀbond with E135 in CDPK1. Unfortunately,
modification of diꢀF meta to the amine in the piperdine was also associated with some
loss of metabolic stability, which likely limited its in vivo efficacy. We did not explore
modification with fluorineꢀdirected modifications at other positions in the piperidine ring,
but conceivably some of these may have a more optimal combination of features.
One of the goals of our study was to obtain molecules with better metabolic stability
to improve in vivo PK properties for treating acute and chronic toxoplasmosis in the
mouse. Compound 24 exhibited many of the desired properties in terms of PK in the
mouse. Although Cmax levels were high, it was relatively rapidly cleared from serum,
likely as a consequence of metabolism in the liver as the halfꢀlife in microsomes was
relatively short. This was partially offset by high oral availability and moderate protein
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binding, resulting in reasonable serum profiles and good AUC profiles. However, we
were only able to obtain free serum levels that exceeded the EC based on free drug
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for a period of ~ 2 hr after oral dosing in the mouse. Nonetheless, dosing schemes of
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5ꢀ50 mg/kg were able to decrease acute infection, preventing weight loss and death in
a majority of immunocompetent animals. Additionally, treatment during acute infection
resulted in dramatically lower cyst counts during chronic infection, likely as a
consequence of decreasing dissemination to the CNS. Indeed, treatment was sufficient
to provide a complete cure in one animal with no evidence of chronic infection.
Although previous studies have largely focused on the acute infection model, the
primary concern for immunocompromised patients is the reactivation of prior chronic
9
ꢀ/ꢀ
infections . Here we modeled this situation using severely immunocompromised Ifngr
37
ꢀ/ꢀ
mice, which are highly susceptible to T. gondii infection . As Ifngr mice completely
lack the ability to respond to IFNγ, a critical mediator of host defense, it is likely that a
single tissue cyst, or single viable organism, is capable of causing lethal infection in this
model. Hence they provide an extremely sensitive measure of the ability of compounds
38
41
to cure infection. Treatment with atovaquone or artemisinin derivatives is able to
prolong survival of such severely immunocompromised mice, but treated animals
ultimately always succumb to infection following removal of these drugs. Similarly,
treatment with sulfadiazine is only able to prevent lethal infection in this model, but not
eliminate the tissue cysts, such that when it is removed, animals quickly succumb due
to reactivation of rapidly growing trachyzoites. Treatment with compound 24 prolonged
ꢀ/ꢀ
survival of Ifngr mice and resulted in a radical cure in some animals, a result not
previously seen with other compounds. The efficacy of compound 24 in this model
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likely stems from its ability to block tachyzoite invasion and egress, but it was also able
to block bradyzoite invasion in vitro, suggesting it may directly affect the formation and
turnover of cysts in vivo. Other PP analogs have been reported to reduce the chronic
cyst burden in infected mice, albeit at very high serum doses, which exceed the level
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where they are selective for CDPK1 . In contrast, compound 24 retains antiꢀ
toxoplasma activity at very modest serum levels, which are well within the range where
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it is selective for CPDK1 in T. gondii
.
CONCLUSIONS
CDPK is an essential kinase in T. gondii and hence has been the focus of development
of potent and selective inhibitors. Here we report new PP inhibitors that were designed
to improve metabolic stability and specificity for CDPK1. The optimal molecule in this
series compound 24 showed low nanomolar inhibition of CDPK1 in vitro, submicromolar
inhibition of parasite growth in vitro, and improved metabolic stability. In vivo
administration of this analog, which showed excellent oral availability and moderate PK
parameters, decreased the severity of acute infection, reduce tissue cyst levels, and
delay reactivation of chronic toxoplasmosis in the mouse. Additionally, compound 24 is
remarkable in being able to completely cure a portion of immunocompromised animals.
As radical cure is currently not possible for humans infected with T. gondii, compounds
that can achieve this goal with higher efficacy would greatly enhance the potential utility
of PP analogs for treatment of toxoplasmosis. Collectively, these advances underscore
the utility of the PP scaffold as a new therapeutic agent for treatment of acute and
chronic toxoplasmosis.
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EXPERIMENTAL SECTION
Chemical synthesis. Starting materials, solvents and reagents obtained commercially
were reagent grade and were used without further purification. NMR spectra were
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obtained at the University of California, San Francisco NMR facility: H NMR were
1
3
recorded on a Bruker AvanceIII HD 400 at 400 MHz and C spectra were recorded on a
Bruker AvanceIII HD 400 at 400 MHz or a Bruker Advance DRX500. Highꢀresolution
mass spectra (HRMS) were acquired by electrospray ionization (ESI) in positive ion
mode using Finnigan LTQ FT mass spectrometer (Thermo) at the QB3/Chemistry Mass
Spectrometry Facility (University of California, Berkeley). Samples were directly injected
into ESI source via syringe pump with flow rate 5 uL/min. Reactions were monitored by
thin layer chromatography (TLC), using Merck silica gel 60 F254 glass plates (0.25 mm
thick). Flash chromatography was conducted with Teledyne Isco RediSep Rf silica flash
cartridges on a Teledyne Isco CombiFlash Rf+. All RPꢀHPLC were performed with a
Waters 2545 binary gradient module equipped with an XBridge prep C18 column using
H O + 0.1% formic acid and CH3CN + 0.1% formic acid (5ꢀ95% gradient) while
2
monitoring at 254 nm. All final compounds were >95% pure as measured by Liquid
Chromatography Mass Spectrometry (LCMS) using a Waters Acquity UPLC/ESIꢀTQD
BEH C18 (1.7 ꢁm) column using H O + 0.1% formic acid and CH3CN + 0.1% formic
2
acid (5ꢀ95% gradient) over 1.8 min at 600 ꢁL/min. Full compound characterization
details are presented in the supporting information. All compounds reported here were
screened for Pan Assay Interference (PAINS) and found to pass all the filters based
contained in the FAFꢀDrugs4 webꢀserver (http://fafdrugs4.mti.univꢀparisꢀdiderot.fr/ ).
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Parasite strains and cell lines. Tachyzoites of T. gondii strains were grown in
monolayers of human foreskin fibroblasts (HFF) maintained in complete medium
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(DMEM containing 10% FBS, 10 mM glutamine, and 10 ꢀg/ml gentamycin) at 37 C in
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% CO . Following natural egress, tachyzoites were purified in HBSS containing 10
2
mM HEPES, 0.1 mM EGTA and separated from host debris using 3.0 micro
polycarbonate membrane filters, followed by centrifugation at 400g. HFF monolayers
obtained from an anonymous donor were provided by Dr. John Boothroyd’s laboratory
(Stanford University). All strains and host cell lines were determined to be mycoplasma
negative using the eꢀMyco plus kit (Intron Biotechnology).
Enzyme expression and purification. Fullꢀlength T. gondii CDPK1 enzyme was
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expressed with a Cꢀterminal His tag in pET22b(+), as described previously . CDPK1
was expressed in BL21 (DE3)V2RpAcYcꢀLIC+LamP E coli, which contains the LamP
phosphatase. A cDNA fragment encoding human Src (hSrc) kinase domain ranging
from amino acids 254ꢀ536 amino acids was amplified using the primers ScrꢀF
(GATATACATATGCAGACTCAGGGCCTGGC) and ScrꢀR
(GTGGTGCTCGAGGAGGTTCTCCCC), and cloned into pET22b using restriction
enzymes NdeI and XhoI. hSrc containing a Thr gatekeeper residue (equivalent of
Thr338 in the full length sequence of the cꢀSrc allele) was expressed in BL21
(DE3)V2RpAcYcꢀLIC+LamP E coli, which contains the LamP phosphatase. Following
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overnight growth in Terrific Broth at 37 C, the culture was diluted 1:100 and grown for 3
o
hr at 37 C (O.D. 0.6ꢀ0.8), then induced with 0.3 mM IPTG during overnight growth at
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5 C or 30 C. Cells were lysed by sonication and soluble proteins purified using HISꢀ
select Nickel Affinity Gel. Purified proteins were dialyzed (50 mM TrisꢀHCL, pH7.5, 150
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mM NaCl), and stored in 25% glycerol containing 0.5 mM DTT at ꢀ80 C. Protein purity
and concentrations were determined by SDSꢀPAGE and staining with SYPRO Ruby
(Invitrogen).
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Crystallography method. For the purpose of crystallization, recombinant TgCDPK1
was expressed, purified, and set up in crystal trial with inhibitors as previously described
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.
Diffracting crystals were obtained by coꢀcrystalization with compounds 3 (PDB code:
5
W9E), 1 (PDB code: 5W8R), 2 (PDB code: 41H8), 13 (PDB code: 5W80), and 4 (PDB
code: 5W91). Crystallographic data were collected at beam line 19ID of Argonne
National Laboratory’s Advanced Photon Source (http://www.sbc.anl.gov/index.html) and
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processed using HKLꢀ3000 . All structures were solved using Phaser for molecular
replacement and the previously deposited TgCDPK1 coordinates (PDB code: 41H8) as
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a search model. The models were refined using Buster and REFMAC combined
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with iterative manual model building using the molecular graphics program Coot . The
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geometry of all final models was checked using MolProbity . Crystallographic details
and refinement statistics are summarized in Table S1. Atomic coordinates and
experimental data will be released on publication.
Homology modeling. Compound 24 was modeled using the coꢀcrystal structure of
compound 13 (PDB code: 5W9E) in Molecular Operating Environment (MOE) version
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2015.1 by Chemical Computing Group . DiꢀFluorine was added to the piperidine of
compound 13 using the ligand builder to create compound 24 and the structure was
prepared and minimized using LigX with the Amber10:EHT force field (used
Protonate3D for protonation, allowed ASN/GLN/HIS “Flips” in Protonate3D, deleted
water molecules farther than 4.5Å from compound 24 or CDPK1, tethering parameters:
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receptor strength at 5000 to minimize large changes to the CDPK1 backbone, fixed
atoms father than 8Å away from compound 24, hydrogens close to compound 24 not
fixed, and refined compound 24ꢀCDPK complex to an RMS gradient of 0.1kcal/mol/Å).
Enzyme assays and IC s. CDPK1 activity was monitored based on phosphorylation
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of syntideꢀ2 peptide (Callbiochem) that was detected using mAb MSꢀ6E6 (MBL Intl,
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Corp) using an ELISA protocol described previously . Kinase reactions were
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conducted at 30 C for 20 min in 20 mM HEPES, pH 7.5, 10 mM MgCl , 1 mM DTT, 2.5
2
mM CaCl , 0.1 mM EGTA, 0.005% Tween 20. Reactions were conducted using 25 ꢀM
2
ATP (the Km for the enzyme) using 140 ng of kinase per reaction. To determine the
potency of inhibitors, duplicate wells were treated with a range of compound
concentrations from 10 ꢀM to 0.5 nM. Inhibitors were preꢀincubated with the enzyme in
the reaction buffer for 10 min before addition of ATP. Individual IC values were
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determined from two or more independent biological replicates and are reported as
mean values. Src activity was monitored by phosphorylation if the Abltide peptide
(EAIYAAPFAKKK, Enzo Life Sciences) that was detected using a monoclonal antibody
to phosphotyrosine (Sigma) using an ELISA protocol similar to that used for CDPK1
above. Kinase reactions were conducted in 50 mM TrisꢀHCl, pH 7.5, 10 mM MgCl , 2.5
2
nM MnCl , 0.2 mM DTT, 0.5 mM EGTA. Reactions were conducted using 5 ꢀM ATP
2
(the Km for the enzyme) using 75 ng of kinase per reaction. Compounds were tested at
10 ꢀM in duplicate, in two biological replicates, and values are reported as mean ±
S.D. For compounds that showed > 50% inhibition at 10 ꢀM, serial dilutions from 10 ꢀM
to 0.5 nM were tested in duplicate to derive IC values.
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CK1ε (ThermoFisher) activity was monitored based on phosphorylation of
dephosphorylated casein (SigmaꢀAldrich) under the following conditions: 50mM TRIS
(
pH 7.4), 10mM MgCl , 0.4 mg/mL casein, 2.5 mM DTT, 2% DMSO, 0.1 mg/mL BSA, 2
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µM ATP (Kmapp), 1µCi 32PꢀATP (Perkin Elmer), 5 nM CK1ε. Additional activity assays
were done at 100 µM ATP with 10nM kinase. To determine the potency of inhibitors,
duplicate wells were treated with a 10ꢀfold dilution of compound from 200 ꢀM to 200 pM.
Inhibitors were preꢀincubated with the enzyme in the reaction buffer for 15 min before
addition of ATP and 32PꢀATP. Upon completion of the reaction (10 minutes for Kmapp
,
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5 minutes for 100 µM ATP) 3ꢁL was spotted onto P81 paper. The paper was dried for
5 minutes under a heat lamp followed by 1x30second wash and 6x5min washes in 1%
Phosphoric Acid. The paper was dried for 15 minutes under a heat lamp followed by
overnight exposure to a phosphor screen imaged on a Typhoon 9500. Intensities were
quantified using densitometry and IC values were determined from quantification of
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two independent biological replicates of triplicate dose response determined by fitting
the data to a sigmoid function in Prism 7.0 (GraphPad Software) and are reported as
mean values.
Kinome screen. Human kinome profiling was conducted using the ThermoFisher
Scientific SelectScreen ACCESS program to profile 489 kinases utilizing three assay
formats: Z’ꢀLYTE (284 kinases), Adapta (39 kinases), and LanthaScreen (166 kinases).
Z’ꢀLYTE and Adapta yield % inhibition values based on the mean of 2 points that
measure the ability of the compound to inhibit an active kinase. LanthaScreen yields %
displacement values based on the mean of 2 points that measure the ability of the
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inhibitor to displace a tracer bound to an inactive or weakly active kinase. Inhibitors
were screened at 1 ꢀM.
Parasite growth assays and EC s. Parasite growth inhibition assays were conducted
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using the type I RH strain, 2F clone that expresses bacterial βꢀgalactosidase (βꢀgal), as
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described previously . Compounds dissolved in DMSO at 10 mM stocks were diluted
in medium to two times final concentrations and added to an equal volume of medium
2
containing 5 x 10 parasites and incubated for 20 min. Mixtures of compounds (ranging
from 10 ꢀM to 0.01 nM) containing 0.1% (v/v) DMSO, or DMSO alone were added to
monolayers of HFF cells grown in 96 well plates, centrifuged at 300 g for 5 min, and
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returned to culture at 37 C, 5% CO . After 4 hr, the plates were washed to remove
2
extracellular parasites and compounds, and then returned to culture for 72 hr. At the
end of the incubation period, the monolayer was lysed in 1% Triton Xꢀ100 and βꢀgal
activity monitored using 1 mM chlorophenol redꢀβꢀDꢀgalactopyranoside by absorption at
23
570 nm, as described previously . Individual EC values were determined from three
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or more independent biological replicates and are reported as mean values.
Host toxicity assays. HFF cells were plated in complete medium and allowed to
o
adhere for 4 hr at 37 C, 5% CO . Compounds diluted to 10 ꢀM in 0.1% (v/v) DMSO, or
2
DMSO alone, were then added and cells were incubated for an additional 72 hr.
Following the incubation period, samples were evaluated using a cell proliferation
assay (CellTiter 96 Aqueous NonꢀRadioactive Cell Proliferation Assay, Promega
Corporation). As a positive control, mitomycin C (10 ꢀM) was included in each
plate. Compounds were tested in duplicate, in two biological replicates, and values are
reported as means ± S.D.
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In vitro analysis of uptake, efflux, metabolism, and protein binding
Assays to monitor uptake and efflux of compounds by polarized epithelial cells were
conduced by Absorption Systems Inc. (Exton, PA, USA). In brief, Cacoꢀ2 cells (Clone
C2BBel from ATCC) were grown to confluence on collagenꢀcoated polycarbonate
membranes on transwell plates. Permeability assays were conducted in Hank’s
balanced salt solution, containing 10 mM HEPES, 10 mM glucose, pH 7.4. Compounds
were added at 5 ꢀM to the apical (A) vs. basolateral (B) side and duplicate samples
taken at 120 min. Samples were analyzed by LCꢀMS/MS and expressed as apparent
permeability (Papp) for uptake (A to B) vs. efflux (B to A) and efflux ration (Papp(B to A) /
Papp(A to B)).
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The stability of compounds in the presence of rat or mouse liver microsomes was
conducted by Absorption Systems, Inc. In brief, rat liver microsomes, or mouse rat liver
microsome were incubated with compounds at 1 ꢀM in a shaking water bath for 60 min.
Aliquots were withdrawn at intervals, extracted, and analyzed by LCꢀMS/MS to evaluate
the remaining parent compound. Halfꢀlives were calculated using a singleꢀphase
exponential decay equation (Graph Pad). Intrinsic clearance was calculated as CL =
int
k/P, where k is the elimination rate constant and P is the protein concentration in the
assay. For plasma protein binding, compounds were incubated at 5 ꢀM in CDꢀ1 mouse
plasma and protein bound fractions were monitored by equilibrium dialysis.
In vivo pharmacokinetic studies
Pharmacokinetic (PK) studies were conducted by Sai Life Sciences Limited (India).
Compounds were administered orally in 50% PEGꢀ400 in PBS, while for i.v.
administration, compounds were dissolved in 20% PEGꢀ400 ꢀPBS. Mean plasma
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concentrations were determined following a single i.v. dose of 3 mg / kg in male Swiss
Albino mice by sampling at 0.05, 0.25, 1, 2, 4 and 8 hrs. Separately, male mice were
used for monitoring mean plasma concentrations following single oral dose 10 /mg / kg
with similar time points. Separate animals were used to determine the brain distribution
relative to plasma levels at 1 hr following a single oral dose of 10 mg/ kg. Selected
compounds were tested for dose response of plasma concentrations following single or
multiple oral administration of a single dose of 10, 25, and 50 mg/ kg at time intervals of
0.25, 1, 2, 4, 8 and 24 hr. Plasma samples were separated processed by precipitation
in acetonitrile and analyzed by LCꢀMS/MS. Pharmacokinetic parameters, including
those for plasma versus brain, and oral bioavailability, were calculated with the nonꢀ
compartmental analysis tools of the Phoenix WinNonLin (v6.3).
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Animal Studies
Animals were obtained from Jackson Laboratories and housed at Washington
University in accordance with the U.S.A. Public Health Service Policy on Humane Care
and Use of Laboratory Animals. Animals were maintained in an Association for
Assessment and Accreditation of Laboratory Animal Care approved facilities and were
approved by the Institutional Animal Studies Committee at the School of Medicine,
Washington University in St. Louis.
Ex vivo bradyzoite treatment assays. Adult (8ꢀ12 weeks old) female CD1 mice were
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infected with tachyzoites of the Pru∆hxg∆ku80; LDH2ꢀGFP (PRUꢀGFP) or
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ME49∆hx::FLUC strains expressing firefly luciferase (FLUC) and allowed to
develop chronic infections. Adult (8ꢀ12 weeks old) CBA/J mice were orally infected with
1
0ꢀ20 tissue cysts of the ME49∆hx::FLUC strain. At 3ꢀ6 weeks postꢀinoculum, brains
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